Halogenated hydrocarbon compounds are high-volume products of the chemical process industry; for example, more than 6 million metric tons of trichloroethylene (TCE), tetrachloroethylene (PCE), trichloroethane, carbon tetrachloride (CT), and chloroform (CF) are produced [in the United States] each year. Those halogenated hydrocarbon compounds most frequently found in groundwater are low molecular weight aliphatic halogenated hydrocarbons: TCE, dichloroethane (DCA), trichloroethane, and PCE. Many of these aliphatic halogenated hydrocarbon compounds, including TCE, have been listed as priority pollutants by the U.S. Environmental Protection Agency, and are known or suspected carcinogens and mutagens. Haloforms (halogenated derivatives of methane) are also frequently detected in groundwaters and drinking waters. Some haloforms are produced during chlorination of water supplies, but inadequate disposal techniques or accidental spillage may also be responsible for the presence of these haloforms.
Several of the halogenated hydrocarbon compounds mentioned above are resistant to biodegradation in aerobic subsurface environments, or their biological transformations are incomplete under anaerobic conditions. For example, under anaerobic conditions, TCE and PCE are known to undergo partial bioconversion to vinyl chloride, a compound which is as much or more of a problem as the original contaminants. Wilson and Wilson, Appl. Env. Microbiol., 49:242-243 (1985).
Current technology for reclaiming groundwater polluted with these halogenated hydrocarbon compounds involves pumping water to the surface and stripping out the contaminants in aeration towers, or removing the pollutants on a sorbent. The former process is not permitted in some states, and the latter is expensive and involves the production of concentrated toxic materials that may present future problems.
In an alternative reclamation method, acetate-degrading methanogenic bacteria have been reported to degrade halogenated hydrocarbon compounds. Chloroform (CF), bromodichloromethane (BDCM), dibromochloromethane (BDCM), bromoform (BF), carbon tetrachloride (CT), 1,1,1-trichloroethane (1,1,1-TCA), 1,1,2,2-tetrachloroethane (1,1,2,2-TECE), and PCE have all been substantially degraded under methanogenic conditions utilizing an anaerobic column with acetate employed as the primary substrate in a medium seeded with a methanogenic mixed bacterial culture. A continuous-flow, fixed-film laboratory scale column operated under these conditions with a 2-day retention time substantially removed these compounds present at column influent concentrations ranging from about 15-40 .mu.g/l. The acclimation period required for significant removal of CF, 1,1,1-TCA, and 1,1,2,2- TECE was about 10 weeks. Bouwer and McCarty, Appl. Env. Microbiol., 45:1286-1294 (1983).
Other anaerobic bacteria are also known to degrade halogenated hydrocarbon-containing compounds. For example, the anaerobic bacteria Methanobacterium thermoautotrophicum and D. autotrophicum have been shown to convert carbon tetrachloride to di- and trichloromethane, and to partially dehalogenate other chlorinated aliphatic compounds. Egli et al., FEMS Microbiol. Letter, 43:257-261 (1987). The above results indicate that the use of methanogenic or other anaerobic bacteria to completely degrade all halogenated hydrocarbons is not commercially viable. These organisms exhibit slow rates of halogenated hydrocarbon destruction, even at low initial concentrations of the hydrocarbons, and are difficult to work with given that anaerobic conditions are required.
Additionally, chloroform is oxidized at rates of 35 nano-moles per gram of cells per minute by the aerobe Methylococcus capsulatus Bath. Higgins et al., Nature, 286:561-564 (1980); Haber et al., Science, 221:1147-1153 (1983). Similar rates of degradation were observed for other haloforms except for carbon tetrachloride, which was not oxidized by Methylococcus capsulatus Bath. Higgins et al., supra; Haber et al., supra.
Certain methane-oxidizing bacteria are known to degrade chlorinated haloforms and halogenated hydrocarbon compounds. For example, soil columns exposed to a surface mixture of 0.6% natural gas (primarily methane) in air for 3 weeks, and having water containing TCE at an average concentration of 150 .mu.g/l added to the column influent at the end of the 3-week acclimation period, resulted in less than 5% of the applied TCE passing through the soil. Wilson and Wilson, supra. A methane-utilizing mixed culture isolated from a marsh has also recently been shown to completely oxidize TCE, vinyl chloride, vinylidene chloride, and dichloroethylene to carbon dioxide. Fogel et al., Appl. Env. Microbiol., 51:720-724 (1986). However, the rate of TCE degradation reported by Fogel et al. was very slow, approximately 2.5 .mu.moles per hour per gram of cells. Additionally, tetrachloroethylene was not oxidized by the mixed culture.
The above studies indicate that several chlorinated haloforms and halogenated hydrocarbon compounds are degradable by combined aerobic/anaerobic incubation under the proper conditions. However, the real potential of methane-oxidizing bacteria, or methanotrophs, for rapidly biodegrading halogenated hydrocarbon compounds such as TCE has not yet been exploited. For example, when TCE was added to the soil column used by Wilson and Wilson, supra, the soil had previously been acclimated to the natural gas mixture for 3 weeks. Similarly, the acclimation period required for significant removal of 1,1,1-TCA and 1,1,2,2 TECE in the Bouwer and McCarty study was about 10 weeks.
It has been known for some time that obligate methanotrophs derive no energy from metabolism of compounds other than methane. Haber et al., supra; Higgins et al., supra. However, methanotrophs are able to degrade numerous hydrocarbon compounds. The ability of methanotrophs to oxidize a wide range of compounds has been associated with the lack of specificity of methane monooxygenase (MMO), an enzyme produced by methanotrophs. Haber et al., supra; Higgins et al., supra. The MMO system of methanotrophic bacteria catalyzes the cleavage of O.sub.2 and incorporation of one oxygen atom into methane to produce methanol.
The MMO system of methanotrophic bacteria can exist in either a soluble or a particulate (i.e., membrane-bound) form, depending on growth conditions. Burrows et al., J. Gen. Microbiol., 130:3327-3333 (1984), reported that copper availability during the growth of the methanotrophic bacterium Methylosinus trichosporium OB3b (Mt OB3b) determined the intracellular location of its MMO (i.e., whether MMO activity was located in the particulate or the soluble fraction of the bacterium). However, the tendency of methanotrophic bacteria cells to elaborate only the membrane-bound (particulate) form of MMO has been a recurring problem in the purification of soluble MMO in quantity. Fox and Lipscomb, Biochem. and Biophys. Res. Comm., 154:165-170 (1988). Burrows et al., supra, reported that the particulate form of the MMO of Mt OB3b differed from the soluble form of the enzyme in that the particulate MMO was unable to oxidize aromatic or alicyclic hydrocarbon compounds. Both the particulate and soluble forms of the MMO of Mt OB3b were shown to oxidize methane, propene, and various n-alkanes.
To date, however, no one has fully exploited the degradation ability of methanotrophic bacteria, nor in particular the degradation ability of the soluble form of the MMO produced by these bacteria, in order to both rapidly and completely degrade halogenated hydrocarbon compounds. For example, the rates of TCE degradation by methanotrophic bacteria reported thus far are unsatisfactorily slow and thus impractical for commercial use. Rates of TCE degradation reported under optimal conditions barely exceed 100 .mu.moles per hour per gram of cells. Fogel et al., supra; Nelson et al., App. Env. Microbiol., 54: 604-606 (1988); Nelson et al., App. Env. Microbiol., 52: 383-384 (1986). The time course of methanotrophic attack upon TCE reported in past studies suggests that TCE is in some way toxic to the bacteria cells, or to the enzymes functional in TCE degradation.
Accordingly, there is a need for a method to rapidly and completely degrade halogenated hydrocarbon compounds such as TCE by employing the soluble form of MMO, or by employing a methanotrophic bacterium which has been cultured in such a way as to produce the soluble MMO.